Interferon Therapy

ABSTRACT

Interferon therapy is improved by concomitant administration of an agent which minimizes the ability of interferon to up-regulate expression of Programmed Cell Death Protein 1 (also known as CD279).

RELATED APPLICATIONS

This application is a Divisional application of co-ending U. S.application Ser. No. 15/430,456 filed 11 Feb. 2017, which in turnasserts priority from provisional patent filing serial no U.S.62/295,268, filed 15 Feb. 2016, the contents of which are hereincorporated by reference.

FEDERALLY-SPONSORED RESEARCH & DEVELOPMENT

None.

JOINT RESEARCH AGREEMENT

Applicant has research agreements with inter alia M. D. Anderson CancerCenter (Houston, Tex.) and The Mayo Clinic (Rochester, Minn.) for workrelated to this application.

SEQUENCE LISTING

None.

PRIOR PUBLIC DISCLOSURES BY THE/AN INVENTOR

None.

BACKGROUND

Interferon has many clinical benefits. For example, interferon is knownto up-regulate the immune system. It thus is potentially useful forrecruiting the patient's innate immune system to identify and attackcancer cells. Interferon's efficacy as an anti-cancer agent, however,has to date proven wanting. This has been puzzling.

For example, the most effective bladder cancer treatment currentlyapproved in The United States is intra-urethral Bacillus Calmette-Guérinvaccine. The antigenic vaccine is thought to stimulate bladder cells toexpress interferon, which in turn recruits the patient's innate immunesystem to better recognize cancer cell surface antigens and attackcancer cells. In over a third of cases, however, the vaccine isineffective.

Similarly, intravesical instillation of exogenously manufacturedinterferon polypeptide has been tested to treat bladder cancer, but hasbeen found less effective than expected.

I have discovered why, and figured out how to fix it.

BRIEF DESCRIPTION

I have found that interferon (either exogenously administered orexpressed in response to a vaccine or other agent which up-regulatesendogenous expression), in addition to stimulating interferonexpression, also stimulates the expression of Programmed Cell DeathProtein 1, also known as CD279. I have thus identified apreviously-unrecognized adverse side effect of interferon therapy:interferon advantageously stimulates certain aspects of the patient'simmune system, yet also up-regulates expression of Programmed Cell DeathProtein 1. The resulting increase in Programmed Cell Death Protein 1 inturn down-regulates protective T cell function. This impairs theeffectiveness of T cells in identifying and attacking cells bearingcancer cell-surface antigen. Thus, interferon produces two conflictingactions: it both increases immune system activity, yet inhibits theability of the immune system to identify cancer cell-surface antigens.

I thus propose improving interferon therapy by co-administering an agentwhich inhibits the expression of Programmed Cell Death Protein 1. Thiswill enable interferon to more fully achieve its therapeutic potential.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart measuring PD-L1 expression in response to interferonexposure, for the RT112 and SW780 human cell lines. Horizontal axis:interferon amount. Vertical axis: polypeptide expressed.

FIG. 2 is a chart measuring TRAIL expression in response to interferonexposure, for the RT112 and SW780 human cell lines. Horizontal axis:interferon amount. Vertical axis: polypeptide expressed.

FIG. 3 is a chart measuring IRF1 expression in response to interferonexposure, for the RT112 and SW780 human cell lines. Horizontal axis:interferon amount. Vertical axis: polypeptide expressed.

FIG. 4 is a photograph of a PAGE gel showing in vitro dose response toincreasing interferon alpha, in an SW780 human cancer cell line.Horizontal axis: interferon amount. Vertical axis: polypeptideexpressed.

FIG. 5 measures expression in RT112 cells of IRF1, FOXA1 and PD-L1 inresponse to interferon exposure, see Example 2. IRF1 served as aninterferon-stimulated gene control. FOXA1 is an example of a type Iinterferon regulated gene that did not change expression afterinterferon exposure.

FIG. 6 measures expression in UC3 cells of IRF1, FOXA1 and PD-L1 inresponse to interferon exposure, see Example 2. IRF1 served as aninterferon-stimulated gene control. FOXA1 is an example of a type Iinterferon regulated gene that did not change expression afterinterferon exposure.

FIG. 7 measures expression in T24 cells of IRF1, FOXA1 and PD-L1 inresponse to interferon exposure, see Example 2. IRF1 served as aninterferon-stimulated gene control. FOXA1 is an example of a type Iinterferon regulated gene that did not change expression afterinterferon exposure.

FIG. 8 measures expression in UC14 cells of IRF1, FOXA1 and PD-L1 inresponse to interferon exposure, see Example 2. IRF1 served as aninterferon-stimulated gene control. FOXA1 is an example of a type Iinterferon regulated gene that did not change expression afterinterferon exposure.

FIG. 9 is a photograph of a 6-lane PAGE gel. It measures the presence ofPD-L1 polypeptide after exposing BBN972 cells to murine interferon.Lanes are (left to right) 0 (zero), 1×10⁰, 1×10¹, 1×10², 1×10³ and 1×10⁴international units interferon/mL of culture medium.

FIG. 10 is a photograph of a 6-lane PAGE gel. It measures the presenceof PD-L1 polypeptide after exposing MB49 #1 (MB49-luc) cells to murineinterferon. Lanes are (left to right) 0 (zero), 1×10⁰, 1×10¹, 1×10²,1×10³ and 1×10⁴ international units interferon/mL of culture medium.

FIG. 11 is a photograph of a 6-lane PAGE gel. It measures the presenceof actin polypeptide after exposing BBN972 cells to murine interferon.Lanes are (left to right) 0 (zero), 1×10⁰, 1×10¹, 1×10², 1×10³ and 1×10⁴international units interferon/mL of culture medium.

FIG. 12 is a photograph of a 6-lane PAGE gel. It measures the presenceof actin polypeptide after exposing MB49 #1 cells to murine interferon.Lanes are (left to right) 0 (zero), 1×10⁰, 1×10¹, 1×10², 1×10³ and 1×10⁴international units interferon/mL of culture medium.

FIG. 13 measures serum interferon a in mice in response tointra-peritoneal injection of Poly I:C.

FIG. 14 measures serum interferon a in mice in response to intra-tumoralinjection of Poly I:C at 6 hours.

FIG. 15 measures PD-L1 expression intra-tumorally 24 hours after PolyI:C (500 mcg) intra-peritoneal injection.

FIG. 16 shows RNA expression in humans treated with nadofarageneradenovec, a recombinant replication-deficient type 5 adenovirus genetherapy vector carrying a human interferon alpha 2B transgene.

FIG. 17 shows MB49 tumor size vs time, for subcutaneous C57BL6/J tumors(n=5 female mice per group). Treatment is 200 mcg q3 days starting onday 10 after tumor implant. Error bard represent SEM.

FIG. 18 shows a Kaplan-Meyer survival curve for female mice withinoculated tumors, treated with saline (lowermost line), IgG (nexthigher line), anti-PD1 monoclonal antibody (next higher line), Poly I:C(next higher line) and a combination of Poly I:C and anti-PD1 monoclonalantibody (highest line).

FIG. 19 compares normalized (mean+/−SD) radiance over time in male mice.Using a log-rank test, these data show combination therapy superior toIgG control (p=0.06), superior to Poly I:C monotherapy (p=0.32), andsuperior to anti-PD1 monoclonal antibody (p=0.14).

FIG. 20 shows “survival portions,” i.e., data showing the survival ofpropensity to survive over time, in male mice treated per FIG. 19.

DETAILED DESCRIPTION

Interferon Therapy

Interferons are a group of signaling proteins. They are expressed andsecreted by human cells in response to the presence of several antigenicpathogens, e.g., viruses, bacteria and parasites, and also tumor cells.Typically, a virus-infected cell releases interferons, signaling nearbybystander cells to heighten their anti-viral defenses. Interferons alsoactivate immune cells such as natural killer cells and macrophages.Interferons increase expression of major histocompatibility complexantigens, which in turn increases presentation of foreign antigens tothe immune system.

Interferons may be sorted or classified according to the type ofreceptor through which they signal. For humans, interferons are oftenthus sorted into three kinds: Type I (interferons which bind to humanIFN-α/β receptors), Type II (interferons which binds to the human IFN-γreceptor) and Type III (interferons which bind to human IFN-λreceptors).

All interferons share several common effects: they are antiviral agentsand they modulate functions of the immune system. Administration of TypeI IFN has been shown to inhibit tumor growth in experimental animals,but the beneficial action in human tumors has not been widelydocumented. A virus-infected cell releases viral particles that caninfect nearby cells. However, the infected cell can prepare neighboringcells against a potential infection by the virus by releasinginterferons. In response to interferon, cells produce large amounts ofan enzyme known as protein kinase R (PKR). This enzyme phosphorylates aprotein known as eIF-2 in response to new viral infections; thephosphorylated eIF-2 forms an inactive complex with another protein,called eIF2B, to reduce protein synthesis within the cell. Anothercellular enzyme, RNAse L—also induced by interferon action-destroys RNAwithin the cells to further reduce protein synthesis of both viral andhost genes. Inhibited protein synthesis destroys both the virus andinfected host cells. In addition, interferons induce production ofhundreds of other proteins-known collectively as interferon-stimulatedgenes (ISGs)—that have roles in combating viruses and other actionsproduced by interferon. They also limit viral spread by increasing p53activity, which kills virus-infected cells by promoting apoptosis. Theeffect of IFN on p53 is also linked to its protective role againstcertain cancers.

Another function of interferons is to up-regulate expression of majorhistocompatibility complex molecules, MHC I and MHC II, and increaseimmune-proteasome activity. Higher MHC I expression increasespresentation of viral peptides to cytotoxic T cells, while theimmune-proteasome processes viral peptides for loading onto the MHC Imolecule, thereby increasing the recognition and killing of infectedcells. Higher MHC II expression increases presentation of viral peptidesto helper T cells; these cells release cytokines (such as moreinterferons and interleukins, among others) that signal to andco-ordinate the activity of other immune cells.

Production of interferons occurs mainly in response to microbes, such asviruses and bacteria, and their products. Binding of molecules uniquelyfound in microbes-viral glycoprotein, viral RNA, bacterial endotoxin(lipopolysaccharide), bacterial flagella, CpG motifs—by patternrecognition receptors, such as membrane bound Toll like receptors or thecytoplasmic receptors RIG-I or MDA5, can trigger release of IFNs. TollLike Receptor 3 (TLR3) is important for inducing interferons in responseto the presence of double-stranded RNA viruses; the ligand for thisreceptor is double-stranded RNA (dsRNA). After binding dsRNA, thisreceptor activates the transcription factors IRF3 and NF-kB, which areimportant for initiating synthesis of many inflammatory proteins. RNAinterference technology tools such as siRNA or vector-based reagents caneither silence or stimulate interferon pathways. Release of IFN fromcells (specifically IFN in lymphoid cells) is also induced by mitogens.Other cytokines, such as interleukin 1, interleukin 2, interleukin-12,tumor necrosis factor and colony-stimulating factor, can also enhanceinterferon production.

Interferon therapy is used (in combination with chemotherapy andradiation) as a treatment for some cancers. This treatment can be usedin hematological malignancy; leukemia and lymphomas including hairy cellleukemia, chronic myeloid leukemia, nodular lymphoma, and cutaneousT-cell lymphoma. Patients with recurrent melanomas receive recombinantIFN-a2b. Both hepatitis B and hepatitis C are treated with IFN-b, oftenin combination with other antiviral drugs. Some of those treated withinterferon have a sustained virological response and can eliminatehepatitis virus. The most harmful strain-hepatitis C genotype Ivirus—can be treated with a 60-80% success rate with the currentstandard-of-care treatment of interferon, RIBAVIRIN™ and recentlyapproved protease inhibitors such as Telaprevir (Incivek™) May 2011,Boceprevir (VICTRELIS™) May 2011 or the nucleotide analog polymeraseinhibitor Sofosbuvir (SOVALDI™) December 2013. Biopsies of patientsgiven the treatment show reductions in liver damage and cirrhosis. Someevidence shows giving interferon immediately following infection canprevent chronic hepatitis C, although diagnosis early in infection isdifficult since physical symptoms are sparse in early hepatitis Cinfection. Control of chronic hepatitis C by IFN is associated withreduced hepato-cellular carcinoma.

The art teaches interferon may be administered as an exogenouspolypeptide.

Alternatively, one may induce endogenous expression of native interferongenes. For example, the art teaches e.g., antigenic BacillusCalmette-Guerin or Mycobacterium or Adenovirus vaccines. Such antigenicpreparations induce the patient's own cells to express interferon.

Alternatively, one may induce endogenous expression of a non-nativeinterferon transgene by transfecting a host cell with a vectordelivering the interferon transgene. Indeed, evenexogenously-administered interferon polypeptide itself acts as amessenger to stimulate interferon production.

As used herein, the term “interferon” (abbreviated “IFN”) referscollectively to type 1 and type 2 interferons including deletion,insertion, or substitution variants thereof, biologically activefragments, and allelic forms. As used herein, the term interferon(abbreviated “IFN”) refers collectively to type 1 and type 2interferons. Type 1 interferon includes interferons-α, -β and -ω andtheir subtypes. Human interferon-α has at least 14 identified subtypeswhile interferon-β has 3 identified subtypes. Particularly, preferredinterferon-alphas include human interferon alpha subtypes including, butnot limited to, α-1 (GenBank Accession Number NP 076918), α-1b (GenBankAccession Number AAL35223), α-2, α-2a (GenBank Accession NumberNP000596), α-2b (GenBank Accession Number AAP20099), α-4 (GenBankAccession Number NP066546), α-4b (GenBank Accession Number CAA26701),α-5 (GenBank Accession Numbers NP 002160 and CAA26702), α-6 (GenBankAccession Number CAA26704), α-7 (GenBank Accession Numbers NP 066401 andCAA 26706), α-8 (GenBank Accession Numbers NP002161 and CAA 26903), α-10(GenBank Accession Number NP 002162), α-13 (GenBank Accession Numbers NP008831 and CAA 53538), α-14 (GenBank Accession Numbers NP 002163 and CAA26705), α-16 (GenBank Accession Numbers NP 002164 and CAA 26703), α-17(GenBank Accession Number NP 067091), α-21 (GenBank Accession NumbersP01568 and NP002166), and consensus interferons as described inStabinsky, U.S. Pat. No. 5,541,293, issued Jul. 30, 1996, Stabinsky,U.S. Pat. No. 4,897,471, issued Jan. 30, 1990, and Stabinsky, U.S. Pat.No. 4,695,629, issued Sep. 22, 1987, the teachings of which are hereinincorporated by reference, and hybrid interferons as described inGoeddel et al., U.S. Pat. No. 4,414,150, issued Nov. 8, 1983, theteachings of which are herein incorporated by reference. Type 2interferons are referred to as interferon γ (EP 77,670A and EP 146,354A)and subtypes. Human interferon gamma has at least 5 identified subtypes,including interferon omega 1 (GenBank Accession Number NP 002168).Construction of DNA sequences encoding inteferons for expression may beaccomplished by conventional recombinant DNA techniques based on thewell-known amino acid sequences referenced above and as described inGoeddel et al., U.S. Pat. No. 6,482,613, issued Nov. 19, 2002, theteachings of which are herein incorporated by reference.

“Biologically active” fragments of interferons may be identified ashaving any antitumor or anti-proliferative activity as measured bytechniques well known in the art (see, for example, Openakker et al.,supra; Mossman, J. Immunol. Methods, 65:55 (1983) and activate IFNresponsive genes through IFN receptor mediated mechanisms. Soluble IFN-αand IFN-β proteins are generally identified as associating with the Type1 IFN receptor complex (GenBank Accession Number NP 000865) and activatesimilar intracellular signaling pathways. IFN-γ is generally identifiedas associating with the type II IFN receptor. Ligand-induced associationof both types of IFN receptors results in the phosphorylation of thereceptors by Janus kinases subsequently activating STATs (signaltransducers and activators of transcription) proteins and additionalphosphorylation events that lead to the formation of IFN-inducibletranscription factors that bind to IFN response elements presented inIFN-inducible genes. Polypeptides identified as activating the IFNpathways following association with Type 1 and/or Type 2 IFN receptorsare considered interferons for purposes of our invention.

Programmed Cell Death Protein 1

Programmed Cell Death Protein 1 (“PD-1”), also known as CD279, is aprotein that in humans is encoded by the PDCD1 gene. PD-1 belongs to theimmunoglobulin superfamily and functions as a cell surface receptor,binding to two known ligands, PD-L1 and PD-L2.

PD-1 plays an important role in down-regulating the human immune systemby preventing the activation of T cells, which in turn reducesautoimmunity and promotes “self-tolerance.” The immune regulatory effectof PD-1 is effected by culling active T cells while protectingsuppressor T cells. PD-1 promotes apoptosis of antigen-specific T cellsin lymph nodes, yet reduces apoptosis in regulatory (“suppressor”) Tcells.

PD-L1 can be highly expressed in certain tumors. This leads to reducedproliferation of, or even elimination of, immune cells in the tumor,impairing the ability of the patient's innate immune system to recognizecancer cell-surface antigen and combat the cancer cells so identified.

PD-1 is expressed on T cells and pro-B cells. PD-1, functioning as animmune checkpoint, plays an important role in down regulating the immunesystem by preventing the activation of T-cells, which in turn reducesautoimmunity and promotes self-tolerance. The inhibitory effect of PD-1is accomplished through a dual mechanism of promoting apoptosis(programmed cell death) in antigen specific T-cells in lymph nodes whilesimultaneously reducing apoptosis in regulatory T cells (suppressor Tcells).

Programmed death 1 is a type I membrane protein of 268 amino acids. PD-1is a member of the extended CD28/CTLA-4 family of T cell regulators. Theprotein's structure includes an extracellular IgV domain followed by atrans-membrane region and an intracellular tail. The intracellular tailcontains two phosphorylation sites located in an immune-receptortyrosine-based inhibitory motif and an immune-receptor tyrosine-basedswitch motif, which suggests that PD-1 negatively regulates TCR signals.This is consistent with binding of SHP-1 and SHP-2 phosphatases to thecytoplasmic tail of PD-1 upon ligand binding. In addition, PD-1 ligationup-regulates E3-ubiquitin ligases CBL-b and c-CBL that trigger T cellreceptor down-modulation. PD-1 is expressed on the surface of activatedT cells, B cells, and macrophages, suggesting that compared to CTLA-4,PD-1 more broadly negatively regulates immune responses.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7family. PD-L1 protein is up-regulated on macrophages and dendritic cells(DC) in response to LPS and GM-CSF treatment, and on T cells and B cellsupon TCR and B cell receptor signaling, whereas in resting mice, PD-L1mRNA can be detected in the heart, lung, thymus, spleen, and kidney.

Monoclonal antibodies targeting PD-1 that boost the immune system arebeing developed for the treatment of cancer. Many tumor cells expressPD-L1, an immunosuppressive PD-1 ligand; inhibition of the interactionbetween PD-1 and PD-L1 can enhance T-cell responses in vitro and mediatepreclinical antitumor activity. This is known as immune checkpointblockade.

One such anti-PD-1 antibody drug, nivolumab, (OPDIVO™, commerciallyavailable from Bristol Myers Squibb Co., Princeton, N.J.), producedcomplete or partial responses in non-small-cell lung cancer, melanoma,and renal-cell cancer, in a clinical trial with a total of 296 patients.Colon and pancreatic cancer patients did not have a response. Nivolumab(OPDIVO™, Bristol-Myers Squibb), which also targets PD-1 receptors, wasapproved in Japan in July 2014 and by the US FDA in December 2014 totreat metastatic melanoma.

Pembrolizumab (KEYTRUDA™ or MK-3475, commercially available from Merck &Co., Rahway, N.J.), which also targets PD-1 receptors, was approved bythe FDA in September 2014 to treat metastatic melanoma. Pembrolizumabhas been made accessible to advanced melanoma patients in the UK via UKEarly Access to Medicines Scheme (EAMS) in March 2015. It is being usedin clinical trials in the US for lung cancer, lymphoma, andmesothelioma. It has had measured success, with little side effects. OnOct. 2, 2015 Pembrolizumab was approved by FDA for advanced (metastatic)non-small cell lung cancer (NSCLC) patients whose disease has progressedafter other treatments.

Other drugs in early stage development targeting PD-1 receptors (oftenreferred to as “checkpoint inhibitors”): Pidilizumab (CT-011, CureTech), BMS 936559 (Bristol Myers Squibb), MPDL328OA (Roche), andatezolizumab (Amgen).

Combination Therapy

I have found that treatment of cancer with interferon—either byadministering interferon polypeptide, or by administering an agent whichinduces cells to express interferon—concomitantly induces expression ofPD-1.

I thus propose improving the efficacy of interferon-based cancer therapyby co-administering interferon with a compound which inhibits theactivity of PD-1.

This entails, for example, administering interferon polypeptideintravenously in an amount effective as cancer therapy, andadministering a monoclonal antibody checkpoint blockade inhibitorintravenously in an amount effective to prevent an interferon-causedincrease in PD-1 expression, and preferably in an amount to reduce theeffect of PD-1.

Alternatively, this entails instilling intravesically an agent whichinduces interferon expression, in an amount effective as cancer therapy,and prophylactically administering a checkpoint blockade inhibitorintravenously in an amount effective to prevent an interferon-causedincrease in PD-1 expression, and preferably in an amount to reduce theeffect of PD-1. The agent can be an antigenic vaccine (such as a virus,or BCG vaccine or Mycobacterium vaccine) which induces interferonexpression. Alternatively, the agent can be a transgene vector whichtransforms a host cell with an expressible interferon transgene.Alternatively, this can be an antigenic virus or bacteria which alsodelivers an interferon transgene.

Example 1—IFNα Induces PD-L1 and TRAIL Expression

Interferon-alpha (IFNa) has not been notably effective clinically. Iposited that this might be more effective in the setting ofvector-mediated IFNa gene therapy. Several years ago, Ibegan a phase IIhuman clinical trial of nadofaragene radenovec (adenovirusvector-mediated interferon alpha 2b). In this experiment, I had measuredthe expression of PD-L1, TRAIL, IRF1 and Lamin A in response to exposureto interferon.

Materials & Methods: RT112 and SW780 cells were cultured in media andthen exposed to media containing interferon alpha polypeptide. Theamount of interferon ranged from zero (control) to 10⁴ internationalunits/mL. Gene expression was evaluated by Western blot and quantitativereal-time PCR using commercially available antibodies and primers. RNAwas isolated from cells in culture with the MIRVANA™ kit (ThermoFisher). mIRs were profiled in RT112 using TAQMAN™ Array Cards (A and B)(Thermo Fisher). Whole genome mRNA expression profiling was performed inRT112 and UC3 with Illumina HumanHT_12_v4 BEADCHIP™ arrays (47323probes).

Results: Results are provided in FIG. 1 to 4. In response to exposure tointerferon, both cell lines up-regulated PD-L1, TRAIL and IRF1expression, and had no measurable effect on Lamin A expression. ForPD-L1, TRAIL and IRF1 expression, the effect was of different magnitudein the different cell lines. See FIG. 1, 2 3, 4.

Conclusions: In a panel of cancer cell lines, interferon exposure leadto significant increases in PD-L1 immune checkpoint expression. I foundthis finding surprising because it implied the reason for the failureto-date of the art to use interferon as an effective cancer therapy.While interferon should theoretically be an effective anti-cancer agent,interferon may also up-regulate expression of PD-L1, thus frustratinginterferon's therapeutic effect.

Example 2—IFNα Induces PD-L1 Expression in a Dose-Dependent Manner

Here I had measured the expression of immune checkpoint PD-L1, micro-RNA(miR) and mRNA expression profiles after treatment with interferonalpha.

Materials & Methods: RT112, T24, UC3, and UC14 cells were cultured inmedia and then exposed for 6 hours to either control media, or mediacontaining 1000 IU/ml of interferon alpha polypeptide. Expression ofPD-L1 was evaluated by Western blot and quantitative real-time PCR usingcommercially available antibodies and primers. RNA was isolated fromcells in culture with the MIRVANA™ kit (Thermo Fisher). mIRs wereprofiled in RT112 using TAQMAN™ Array Cards (A and B) (Thermo Fisher).Whole genome mRNA expression profiling was performed in RT112 and UC3with Illumina HumanHT_12_v4 BEADCHIP™ arrays (47323 probes). Allexperiments were performed in triplicate to increase statisticalreliability.

Results: All cell lines up-regulated the expression PD-L1 in response toexposure to IFNa. This effect was most pronounced in RT112 cells, seeFIG. 5, than in UC3 cells, FIG. 6, In contrast, the expression of threepotential oncomIR regions was significantly down-regulated afterexposure to IFNa in RT112:1233 cells (p=0.0036), 19b-1# (p=0.0157), and222# (p=0.0061). Analyzing differentially-expressed genes with at least2-fold differences in log (expression) (false discovery rate <0.001)after IFNa exposure, there were 302 and 181 differentially expressedgenes in the RT112 and UC3 cell lines, respectively. Top-rankedIFNa-induced genes in both cell lines included several that had not beenpreviously described in bladder cancer, including IFIT2 (negativeregulator of metastasis) and IFI27 (associated with sensitivity toTRAIL). IFNa-induced PD-L1 expression was also demonstrable on the mRNAgene chip with fold-changes paralleling real-time PCR data.

Conclusions: In a panel of cancer cell lines, IFNa exposure lead tosignificant increases in PD-L1 immune checkpoint expression. Array-basedmicroRNA and mRNA profiling revealed novel potential mediators of IFNaresponse in bladder cancer. This bladder IFNa profile may be useful asan intermediate endpoint to measure response to adenoviral IFNa genetherapy. Future prediction of PD-L1 expression with IFNa therapy maylead to rational combination treatments utilizing immune checkpointinhibitors.

Example 3—Murine Interferon Induces PD-L1 Expression

Materials and Methods: BBN972 and MB49 #1 (MB49-luc) cells werecultured, and then exposed to media containing from 0 (zero) to 1×10⁴international units of murine interferon. Subsequent expression of PD-L1and (as a control) actin were measured.

Results: Murine interferon had no effect on the expression of actin ineither cell line. See FIGS. 11, 12. In contrast, Murine interferon had amarked, dose-dependent effect on PD-L1 expression. See FIGS. 9, 10.

Conclusions: These data show that the effect of interferon on PD-L1expression is not limited to human interferon alpha 2a, nor indeed tohuman interferon. Rather, the effect of interferon on expression ofPD-L1 appears to be generic to interferon generally.

Example 4—Polyinosinic:Polycytidylic Acid (Poly I:C) Induces PD-L1

Materials & Methods: The foregoing data indicate that interferon inducesPD-L1 expression, does so in a dose-dependent manner, does so quickly,and does so apparently in response to interferon from different species.Given the effect regardless of the animal species from which theinterferon was taken, I hypothesized that the effect might not belimited to interferon, and might be more generally provoked by immunestimulants of other types. To test the concept, I had evaluatedPolyinosinic:polycytidylic acid (often abbreviated “poly I:C”). Poly I:Cis an immune-stimulant. It is used in the form of its sodium salt tosimulate viral infections. Poly I:C is structurally similar todouble-stranded RNA. dsRNA is present in some viruses. I had Poly I:Cadministered via intra-peritoneal injection to laboratory mice withimplanted plc or ulc tumors.

Results. FIG. 13 shows that control mice (n=3) showed a de minimusbaseline measure of serum interferon a. In contrast, intra-peritonealinjection of Poly I:C produces a time-dependent increase in seruminterferon a. FIG. 14 shows results of intra-tumoral injection of PolyI:C at 6 hours. The data (n=1 for each series) show that intra-tumorinterferon a increases significantly in plc tumors, increases somewhatin ulc tumors, and does not measurably increase in control tumors. FIG.15 shows that Poly I:C (500 mcg) also induces (at 24 hours) PD-L1expression intra-tumorally (Mann Whitney p=0.0495).

Conclusions: These data indicate that PD-L1 expression is induced notmerely by interferon, but by Poly I:C, a compound which mimics dsRNA andwhich induces interferon expression.

Example 5—Interferon Viral Gene Therapy Induces PD-L1 in Humans

Materials & Methods: These data are taken from a human Phase II humanclinical trial for nadofaragene radenovec replication-deficientadenoviral gene therapy vector carrying a human interferon alpha 2btransgene in patients unresponsive to or refractory after BCG therapy.That study plan has been published and is incorporated here byreference.

Results: FIG. 16 shows RNA expression in eight (8) treatment cycles inhumans treated with nadofaragene radenovec recombinantreplication-deficient adenovirus gene therapy vector carrying a humaninterferon alpha 2B transgene. Odd (white color coded) columns measureRNA transcription before treatment; even (light blue color coded)columns measure after. RNA amounts are shown quantitatively, light greenshowing the least and light red the most. Columns 1 and 2 show PD-L1 RNAincreasing from −2 before treatment to +2 after. Columns 3 and 4similarly show PD-L1 RNA increasing from −2 before treatment to +3after. In all, one third of the treatment pair show a significantincrease in PD-L1 expression after treatment. Treatment alsoup-regulated other immune checkpoint markers.

Conclusions: These data show that one third of patients demonstrateinduction of T-cell and immune checkpoint markers (including PD-L1)after treatment with interferon gene therapy.

Example 6—Combination Therapy Increases Survival

Materials & Methods: Female laboratory rats were inoculated with tumorcells, and the cells allowed to develop into measurable tumors. The ratswere then treated with saline (control), IgG (as a control), anti-PD1monoclonal antibody (monotherapy), Poly I:C (monotherapy to induceinterferon expression) and a combination of Poly I:C and anti-PD1monoclonal antibody (combination therapy).

Results: FIG. 17 shows MB49 tumor size vs time, for subcutaneousC57BL6/J tumors (n=5 female mice per group). Treatment is 200 mcg q3days starting on day 10 after tumor implant. Error bard represent SEM.The highest (yellow) line, showing the largest tumor volume at day 40,is control group (all groups n=5, female-only). The next lowest (blue)line is the IgG control. The next lowest (red) line is Poly I:C. Thenext lowest (green) line is anti-PD1 Monoclonal antibody. The lowest(black) line, laying on the X axis itself, is combination therapy.

FIG. 18 shows a Kaplan-Meyer survival curve for female mice withinoculated tumors, treated with saline (lowermost line), IgG (nexthigher line), anti-PD1 monoclonal antibody (next higher line), Poly I:C(next higher line) and a combination of Poly I:C and anti-PD1 monoclonalantibody (highest line). These data show that combining aninterferon-inducing agent (Poly I:C) and a PD1 inhibitor (an anti-PD1monoclonal antibody) increases survival significantly: at 50 days, -20%of control animal remain alive, 50% of Poly I:C animals remain alive,and 100% of combination treated animals remain alive.

FIG. 19 compares normalized (mean+/−SD) radiance over time in male mice.Using a log-rank test, these data show combination therapy superior toIgG control (p=0.06), superior to Poly I:C monotherapy (p=0.32), andsuperior to anti-PD1 monoclonal antibody (p=0.14).

FIG. 20 shows “survival portions,” i.e., data showing the survival ofpropensity to survive, over time.

Conclusions: These data show combination therapy synergisticallyeffective, imparting a more than merely additive effect.

Example 7—Superficial Spreading Melanoma

Materials & Methods: A human patient diagnosed with superficialspreading melanoma is treated by wide local excision and sentinel nodebiopsy to confirm lack of spread of the disease to the lymph system ordistal organs. The patient is then treated with a combination ofnadofaragene radenovec and KEYTRUDA™. Treatment is initiated as soon aspractical after surgical resection.

Nadofaragene radenovec is a replication-deficient, recombinantadenoviral gene therapy vector bearing an interferon alpha 2b transgene.The manufacture of such gene therapy vectors is described in, e.g.,Muralidhara Ramachandra et al., Selectively Replicating Viral Vector,U.S. Pat. No. 7,691,370. The isolation of interferon transgenes isdescribed in e.g., Charles Weissmann, DNA Sequences, Recombinant DNAMolecules and Processes for Producing Human Interferon-LikePolypeptides, U.S. Pat. No. 6,835,557.

KEYTRUDA™ brand pembrolizumab is a humanized monoclonal anti-programmedcell death-1 (PD-1) antibody (IgG4/kappa isotype with a stabilisingsequence alteration in the Fc region).

Nadofaragene radenovec is provided in single-dose vials. One dose ofnadofaragene radenovec is reconstituted in sterile saline for injectionand administered subcutaneously locally to the excision site.Administration is repeated once every four weeks. One vial of KEYTRUDA™powder contains 50 mg of pembrolizumab. KEYTRUDA™ is administered as anintravenous infusion over 30 minutes, repeated every 3 weeks, andpatients are treated until disease progression or unacceptable toxicity.Atypical responses (i.e., an initial transient increase in tumour sizeor small new lesions within the first few months followed by tumourshrinkage) may be observed. It is preferred to continue treatment forclinically-stable patients with initial evidence of disease progressionuntil disease progression is confirmed.

Test subjects are enrolled and then assigned to a treatment group:excision followed by KEYTRUDA™ only, excision followed by nadofarageneradenovec only, excision followed by KEYTRUDA™ and nadofarageneradenovec concomitantly, excision followed by nadofaragene radenovec andNSAID (a COX-2 inhibitor), and excision followed by KEYTRUDA™ andnadofaragene radenovec and NSAID concomitantly.

Results: The primary efficacy outcome measures are progression freesurvival (as assessed by e.g., an Integrated Radiology and OncologyAssessment review using Response Evaluation Criteria in Solid Tumours[RECIST]), overall survival, and sentinel node biopsy. Other efficacyoutcome measures may be overall response rate and response duration.Subsequent sentinel node biopsy is expected to show no spread of thedisease.

I expect that administration of nadofaragene radenovec with COX-2inhibitor will demonstrate superior efficacy to nadofaragene radenoveconly. I expect that administration of KEYTRUDA™ and nadofarageneradenovec concomitantly will demonstrate superior efficacy outcomemeasures as compared to administration of either agent alone, and Iexpect this benefit to be more than merely additive. I expect thatadministration of KEYTRUDA™ and nadofaragene radenovec and NSAIDconcomitantly will demonstrate superior efficacy outcome measures ascompared to administration of KEYTRUDA™ alone or nadofaragene radenovecand NSAID alone, and I expect this benefit to be more than merelyadditive.

Example 8—Superficial Spreading Melanoma

Materials & Methods: KEYTRUDA™ as in the foregoing example.

As a source of interferon, SYLATRON™ PEG-ylated interferon alpha 2b,administered subcutaneously at 6 mcg/kg once weekly for 8 doses(induction), followed by 3 mcg/kg once weekly for up to 5 years(maintenance). If SYLATRON™ dosage modification is required during weeks1-8 of treatment (induction) because of adverse reactions, a 3-stepdecrease from original dosage (6 mcg/kg once weekly) is preferred (i.e.,decrease dosage to 3 mcg/kg once weekly; if needed, decrease to 2 mcg/kgonce weekly; then, if needed, further decrease to 1 mcg/kg once weekly).If dosage modification required during weeks 9-260 of treatment(maintenance) because of adverse reactions, a 2-step decrease fromoriginal dosage (3 mcg/kg once weekly) recommended (i.e., decreasedosage to 2 mcg/kg once weekly; if needed, decrease to 1 mcg/kg onceweekly).

Test subjects are enrolled and then assigned to a treatment group:excision followed by KEYTRUDA™ only, excision followed by SYLATRON™only, excision followed by KEYTRUDA™ and SYLATRON™ concomitantly,excision followed by SYLATRON™ and NSAID (a COX-2 inhibitor), andexcision followed by KEYTRUDA™ and SYLATRON™ and NSAID concomitantly.

Results: The primary efficacy outcome measures are progression freesurvival (as assessed by e.g., an Integrated Radiology and OncologyAssessment review using Response Evaluation Criteria in Solid Tumours[RECIST]), overall survival, and sentinel node biopsy. Other efficacyoutcome measures may be overall response rate and response duration.Subsequent sentinel node biopsy is expected to show no spread of thedisease.

I expect that administration of SYLATRON™ with COX-2 inhibitor willdemonstrate superior efficacy to SYLATRON™ only. I expect thatadministration of KEYTRUDA™ and SYLATRON™ concomitantly will demonstratesuperior efficacy outcome measures as compared to administration ofeither agent alone, and Iexpect this benefit to be more than merelyadditive. I expect that administration of KEYTRUDA™ and SYLATRON™ andNSAID concomitantly will demonstrate superior efficacy outcome measuresas compared to administration of of KEYTRUDA™ alone or SYLATRON™ andNSAID alone, and I expect this benefit to be more than merely additive.

Example 9—Non-Small Cell Lung Cancer

Materials & Methods: Pharmaceutical Agents as per Example 7 above. Humantest subjects are diagnosed as having Non-Small-Cell Lung Carcinoma.Patients are screened according to Greene, Frederick L., Cancer StagingManual (American Joint Committee on Cancer, publ., 6th edition) toassure that comparable test subjects have comparable disease. Testsubjects are screened for treatment based on the tumor expression ofPD-L1, expression confirmed by a validated test.

The recommended dose of KEYTRUDA is: 200 mg for NSCLC that has not beenpreviously treated with chemotherapy, and 2 mg/kg for NSCLC that hasbeen previously treated with chemotherapy or for melanoma.

nadofaragene radenovec is administered by intra-pleaural infusion. Thismethod is illustrated in United States Patent publication US2014/17202at FIG. 2.

Test subjects are enrolled and then assigned to a treatment group:KEYTRUDA™ only, nadofaragene radenovec only, KEYTRUDA™ and nadofarageneradenovec concomitantly, nadofaragene radenovec and NSAID (a COX-2inhibitor), and KEYTRUDA™ and nadofaragene radenovec and NSAIDconcomitantly.

Results: The primary efficacy outcome measures are progression freesurvival, overall survival, and sentinel node biopsy. Other efficacyoutcome measures may be overall response rate and response duration.Subsequent sentinel node biopsy is expected to show no spread of thedisease.

I expect that administration of nadofaragene radenovec with COX-2inhibitor will demonstrate superior efficacy to nadofaragene radenoveconly. I expect that administration of KEYTRUDA™ and nadofarageneradenovec concomitantly will demonstrate superior efficacy outcomemeasures as compared to administration of either agent alone, and Iexpect this benefit to be more than merely additive. I expect thatadministration of KEYTRUDA™ and nadofaragene radenovec and NSAIDconcomitantly will demonstrate superior efficacy outcome measures ascompared to administration of KEYTRUDA™ alone or nadofaragene radenovecand NSAID alone, and I expect this benefit to be more than merelyadditive.

Summary

The above Examples discuss treating certain cancers. Our discovery,however, may be more generally used to treat any condition whichbenefits from interferon signaling, and which suffers fromover-expression of CD279.

In the appended claims, I use the term “treat” not to require completecure, but to ameliorate. For example, “treating” cancer may be achievedby completely eliminating the cancer, and also by, for example, slowingtumor growth, reducing the risk of mortality or slowing diseaseprogression when compared to patients who do not have such treatment.

Given our disclosure here, the artisan can readily see specificapplications or variants of it. For example, while the above discussionmentions specific species of human interferon, other species andinterferon derivatives or analogs which function similarly will providethe same benefit. Thus, I intend the legal coverage of our patent to bedetermined not by the Examples I discuss, but by the appended legalclaims and permissible equivalents thereof.

When the appended legal claims refer to treating at about “the sametime,” see e.g., original claim 3, this requires the two compounds workin the patient at the same time. It does not require contemporaneousadministration. Thus, one could administer the first agent a week afteradministering the second agent, if the effect of the second agentpersists for at least a week.

I claim:
 1. A method for improving the efficacy of interferon as a humantherapeutic by co-administering interferon with an agent which reversesan immune-suppressive effect of interferon, the method comprising: a.administering to a human patient diagnosed with a condition which may betreated with interferon, a first agent which increases the humanpatient's level of interferon and does so in an amount effective totreat the condition; and b. administering to the human patient amonoclonal antibody against programmed cell death protein 1, themonoclonal antibody administered in an amount sufficient to reduce theactivity of programmed cell death protein 1; whereby the monoclonalantibody counteracts the decrease in immune activity caused byinterferon.
 2. The method of claim 1, consisting essentially of: a.administering to a human patient diagnosed with a condition which may betreated with interferon, a first agent which increases the humanpatient's level of interferon and does so in an amount: effective totreat the condition; and b. administering to the human patient amonoclonal antibody against programmed cell death protein 1, themonoclonal antibody administered in an amount sufficient to reduce theactivity of programmed cell death protein 1; whereby the monoclonalantibody counteracts the decrease in immune activity caused byinterferon.
 3. The method of claim 1, wherein the first agent whichincreases the human patient's level of interferon comprisesexogenously-manufactured interferon polypeptide.
 4. The method of claim1, wherein the first agent which increases the human patient's level ofinterferon comprises a gene therapy vector carrying an expressibleinterferon transgene.
 5. The method of claim 4, wherein the gene therapyvector carrying an expressible interferon transgene comprisesnadofaragene radenovec.
 6. The method of claim 1, wherein the firstagent which increases the human patient's level of interferon comprisesan interferon-inducing antigen.
 7. The method of claim 6, wherein theinterferon-inducing antigen comprises poly I:C.